Cytotoxicity Assays

CELLestial™ Kits Trouble Shooting
CELLestial™ Dyes & Kits FAQs

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Blue nuclear counterstain (in the dual color detection reagent) signal is dramatically reduced in treated cells

• If the blue nuclear signal decreases by more than 30%, the testing compound is generally cytotoxic >> optimize the concentration of the perturbing reagent.
  
  

Poor staining observed.

• Stained cells may have been exposed to strong light >> Mito-ID® MP dye is light sensitive; be sure to protect samples from light and analyze them immediately after staining.

Control cells without treatment show low orange signal.

• Control cells are not healthy. Extended storage of cells after staining may adversely affect their health >> Use healthy cells and acquire data soon after staining.
  The appropriate incubation time depends upon the individual cell type and cell concentration used.  Optimize the incubation time for each experiment. DO NOT wash the cells after dye loading.

Cell staining is too strong

• Mitochondrial membrane potential dye is too concentrated for the cell type used >> optimize for different cell types by diluting the staining solution further with assay buffer.

More than two populations appear upon apoptosis induction

• It’s the property of the dye >> apoptosis is a multi-stage process and Nuclear-ID green dye can distinguish different levels of condensation.
  
  

Chromatin condensation is not observed

• Incorrect drug concentration or incubation time for cell type:
• Make sure the cell preparation protocol generates single cells with minimal clumping.  Filtration or triturating of cell suspensions may be required.
• Systematically vary the dye-to-cell ratio.
• Optimize for cell type, medium or buffer used, time of incubation, temperature of incubation.
• Verify that the flow cytometer is correctly aligned, with stable fluidics, using calibrated fluorescent beads of known CV.
• Verify that the cell concentration is 1 x 10⁵ to 1 x 10⁶ cells/mL.
• Lower PMT setting on flow cytometry.
  
  

Healthy cells show very high fluorescence intensity:

• PMT setting is too high >> Lower PMT setting on flow cytometer.
• Incubation temperature >> Incubation with the dye should be at 4°C.
• Permeabilized cells >> The dye gets intercalated in the DNA giving higher fluorescence.